Class I major histocompatibility molecules play a key role in recognition of antigens by CD8+ T lymphoctyes. By binding peptides in the endoplasmic reticulum and transporting them to the cell surface, class I molecules allow T lymphocytes to scan for abnormal protein expression. This allows elimination of cancerous cells or cells infected with intracellular pathogens. The peptide binding clefts of individual class I molecules differ drastically in shape and peptide content. Such diversity allows T lymphocytes to survey a wide variety of antigens, and suggests that specificity of peptide binding is controlled largely by the shape of the class I binding cleft. How peptides bind to class I molecules in vivo is presently not well understood, and interactions with accessory proteins influence the process. How several accessory proteins influence the biogenesis and peptide binding properties of class I molecules within cells, with the eventual goal of manipulating antigen presentation will be investigated. 1) The effect of polymorphism in class I molecules on binding to calnexin and calreticulin will be examined. The investigators previously showed that two human class I proteins, encoded by A*0201 and B*0702, bind weakly and strongly to calnexin respectively. A panel of sixteen additional HLA-A, -B and -C heavy chains will be studied to determine if patterns of binding can be discerned, and whether strong and weak binders have different kinetics of transport. 2) The position dependence of the glycan on class I heavy chains for binding calnexin and calreticulin will be tested. Novel glycan acceptor sites will be introduced by site directed mutagenesis into class I heavy chains carrying a mutation which prevents their glycosylation and binding to calnexin. 3) How class I molecules bind to TAP/tapasin involved in transporting peptides into the endoplasmic reticulum will be determined. Potential sites of interaction in A*0201 and B*0702 include parts of the a2 and a3 domains, as well as the peptide binding cleft. 4) Regions of calnexin important for ligand binding will be identified by mutagenesis followed by transfection of mutant clones into the calnexin negative human cell, NKR. Several assays have been established to determine whether calnexin mutants are functional.